Micronucleated CD71-positive reticulocytes: a blood-based endpoint of cytogenetic damage in humans

The frequency of micronuclei (also known as Howell-Jolly bodies) in peripheral blood erythrocytes of humans is extremely low due to the efficiency with which the spleen sequesters and destroys these aberrant cells. In the past, this has precluded erythrocyte-based analyses from effectively measuring chromosome damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronucleated reticulocytes (MN-RET) in human blood. Differential staining of these cells was accomplished by combining the immunochemical reagent anti-CD71-FITC with a nucleic acid dye (propidium iodide plus RNase). The immunochemical reagent anti-CD42b-PE was also incorporated into the procedure in order to exclude platelets which can interfere with analysis. This analytical system was evaluated with blood samples from ten healthy volunteers, one splenectomized subject, as well as samples collected from nine cancer patients before and over the course of radio- or chemotherapy. The mean frequency of MN-RET observed for the healthy subjects was 0.09%. This value is nearly two orders of magnitude higher than frequencies observed in mature erythrocytes, and is approximately half the MN-RET frequency observed for the splenectomized subject (0.20%). This suggests that the spleen's effect on micronucleated cell incidence can be minimized by restricting analyses to the youngest (CD71 -positive) fraction of reticulocytes. Furthermore, MN-RET frequencies were significantly elevated in patients undergoing cancer therapy. Collectively, these data establish that micronuclei can be quantified in human peripheral blood reticulocytes with a single-laser flow cytometer, and that these measurements reflect the level of chromosome damage which has occurred in red marrow space. (C) 2003 Elsevier B.V. All rights reserved.